This application is the national phase under 35 U.S.C. xc2xa7371 of prior PCT International Application No. PCT/JP98/00858 which has an International filing date of Mar. 2, 1998 which designated the United States of America, the entire contents of which are hereby incorporated by reference.
The present invention relates to a DNA fragment containing at least one gene concerned in biotin biosynthesis and utilization thereof.
Biotin is an essential vitamin for human beings, animals, plants and some microorganisms and is useful as a food additive for human beings or animals. As a process for producing biotin by using a microorganism, there have been known a process using a streptomyces or a micro-monospore (JP-B-41-21756), a process using a sporobolomyces (JP-B-42-3074), a process using a bacillus, a chromo-bacterium or a pseudomonas (JP-A-56-160998), a process using a sphingomonas (JP-A-6-133790), etc. There have been also proposed processes for breeding a microorganism in which a gene concerned in biotin biosynthesis and isolated from a microorganism capable of producing biotin is introduced into another microorganism by a genetic engineering technique to promote the expression of the gene concerned in biotin biosynthesis, whereby the activity of an enzyme capable of catalyzing biotin biosynthesis reaction is increased to improve the biotin productivity (JP-A-61-202686, JP-A-2-27980, JP-A-7-231789, etc.).
As genes concerned in biotin biosynthesis in microorganism cells, there are known bio A, bio B, bio F, bio D, bio C and bio H genes derived from Escherichia coli (Journal of Biological Chemistry, voL. 263, 19577-19585(1988)). The bio A gene codes for an enzyme having 7,8-diaminopelargonate aminotransferase activity. The bio B gene codes for an enzyme having biotin synthase activity. The bio F gene codes for an enzyme having 7-keto-8-aminopelargonate synthetase activity. The bio D gene codes for an enzyme having desthiobiotin synthetase activity. The bio C gene participates in a biosynthesis stage upstream to pimelyl Co-A in the biotin biosynthetic pathway. The action of the bio H gene is not clear. In the biosynthetic pathway in Escherichia coli, intracellular pimelyl Co-A is converted to 7-keto-8-aminopelargonic acid by 7-keto-8-aminopelargonate synthetase, this 7-keto-8-aminopelargonic acid is converted to diaminopelargonic acid by 7,8-diaminopelargonate aminotransferase, this diaminopelargonic acid is converted to desthiobiotin by desthiobiotin synthetase, this desthiobiotin is converted to biotin by biotin synthase, whereby biotin is synthesized. When the bio C gene is deleted, the amount of biotin produced is decreased. Therefore, it is considered that the bio C gene codes for an enzyme having activity to catalyze a reaction in a biosynthesis stage upstream to pimelyl Co-A (xe2x80x9cFermentation and Industryxe2x80x9d, 46, 102-111(1988)). The base sequences of the bio A, bio B, bio F, bio D, bio C and bio H genes derived from Escherichia coli have already been specified. It is known that the bio A, bio B, bio F, bio D and bio C genes form an operon the transcription of which is controlled by an operator.
As genes concerned in biotin biosynthesis and derived from microorganisms belonging to genera other than the genus Escherichia, there have been reported genes derived from Serratia marcescens (GenBank database, accession No. D17468) and genes derived from Bacillus subtilis (JP-A-7-231789). The base sequence of each of these genes has been specified. It is known that although the base sequences of these genes are different from those of the genes of Escherichia coli, the functions of gene products and the biosynthetic pathway of biotin in the case of the former genes are substantially the same as in the case of the latter genes (Escherichia coli). On the other hand, genes concerned in biotin biosynthesis and derived from Bacillus sphaericus have been reported (Ohsawa et al., Gene 80, 39-48(1989), Gloeckler et al., Gene 87, 63-70(1990)). The genes of Bacillus sphaericus are different from those of Escherichia coli in the following respects: genes concerned in a biosynthesis stage upstream to pimelyl Co-A, the order and cluster formation of bio genes, etc. (Gloeckler et al., Gene 87, 63-70(1990)).
However, as to genes concerned in biotin biosynthesis and derived from a microorganism belonging to the genus Sphingomonas, their base sequences and either the functions or the structure of gene products have not been known at all. Therefore, there have been no technique for utilizing a gene concerned in biotin biosynthesis and derived from a microorganism belonging to the genus Sphingomonas, for breeding of a biotin-producing microorganism by genetic engineering.
In such circumstances, the present inventors earnestly investigated and consequently found that transformants used for producing biotin or a biotin precursor can be prepared by isolating a DNA fragment containing a gene concerned in biotin biosynthesis from a microorganism belonging to the genus Sphingomonas, inserting the DNA fragment to a vector, and then introducing the vector into host cells. Thus, the present invention has been accomplished.
The present invention provides the following:
1) A DNA fragment containing a gene concerned in biotin biosynthesis and derived from a microorganism belonging to the genus Sphingomonas.
2) A DNA fragment according to the above item 1, wherein said gene is selected from the group consisting of a 7-keto-8-aminopelargonate synthetase gene, a 7,8-diaminopelargonate aminotransferase gene, a desthiobiotin synthetase gene, a biotin synthase gene, and a gene coding for an enzyme having activity to catalyze a reaction in a biosynthesis stage upstream to pimelyl Co-A in the biotin biosynthetic pathway.
3) A DNA fragment according to the above item 1, herein said gene is a gene coding for 7-keto-8-aminopelargonate synthetase.
4) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 1 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 1, and having 7-keto-8-aminopelargonate synthetase activity.
5) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 2 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 2, and having 7-keto-8-aminopelargonate synthetase activity.
6) A DNA fragment containing a gene having the base sequence shown as SEQ ID NO: 3, 5or 7.
7) A DNA fragment according to the above item 1, wherein said gene is a gene coding for 7,8-diaminopelargonate aminotransferase.
8) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 9 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 9, and having 7,8-diaminopelargonate aminotransferase activity.
9) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 10 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 10, and having 7,8-diaminopelargonate aminotransferase activity.
10) A DNA fragment containing a gene having the base sequence shown as SEQ ID NO: 11 or 13.
11) A DNA fragment according to the above item 1, wherein said gene is a gene coding for desthiobiotin synthetase.
12) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 15 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 15, and having desthiobiotin synthetase activity.
13) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 16 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 16, and having desthiobiotin synthetase activity.
14) A DNA fragment containing a gene having the base sequence shown as SEQ ID NO: 17 or 19.
15) A DNA fragment according to the above item 1, wherein said gene is a gene coding for biotin synthase.
16) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 21 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 21, and having biotin synthase activity.
17) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 22 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 22, and having biotin synthase activity.
18) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 27, and having biotin synthase activity.
19) A DNA fragment containing a gene having the base sequence shown as SEQ ID NO: 23, 25or 28.
20) A DNA fragment according to the above item 1, wherein said gene is a gene coding for an enzyme having activity to catalyze a reaction in a biosynthesis stage upstream to pimelyl Co-A in the biotin biosynthetic pathway.
21) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 30 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 30, and having activity to catalyze a reaction in a biosynthesis stage upstream to pimelyl Co-A in the biotin biosynthetic pathway.
22) A DNA fragment containing a gene coding for a protein having the amino acid sequence shown as SEQ ID NO: 31 or an amino acid sequence formed by deletion, substitution, modification or addition of one or more amino acids in the amino acid sequence shown as SEQ ID NO: 31, and having activity to catalyze a reaction in a biosynthesis stage upstream to pimelyl Co-A in the biotin biosynthetic pathway.
23) A DNA fragment containing a gene having the base sequence shown as SEQ ID NO: 32 or 34.
24) A DNA fragment having a partial base sequence of a gene concerned in biotin biosynthesis and derived from a microorganism belonging to the genus Sphingomonas.
25) A DNA fragment according to the above item 24, wherein said gene is a 7-keto-8-aminopelargonate synthetase gene, a 7,8-diaminopelargonate aminotransferase gene, a desthiobiotin synthetase gene, a biotin synthase gene, or a gene coding for an enzyme having activity to catalyze a reaction in a biosynthesis stage upstream to pimelyl Co-A in the biotin biosynthetic pathway.
26) A DNA fragment comprising a region coding for a protein of a gene concerned in biotin biosynthesis and derived from a microorganism belonging to the genus Sphingomonas.
27) A DNA fragment according to the above item 26, wherein said gene is a 7-keto-8-aminopelargonate synthetase gene, a 7,8-diaminopelargonate aminotransferase gene, a desthiobiotin synthetase gene, a biotin synthase gene, or a gene coding for an enzyme having activity to catalyze a reaction in a biosynthesis stage upstream to pimelyl Co-A in the biotin biosynthetic pathway.
28) A DNA fragment comprising a gene expression regulatory region of a gene concerned in biotin biosynthesis and derived from a microorganism belonging to the genus Sphingomonas, said region being upstream to a region coding for a protein.
29) A DNA fragment according to the above item 28, wherein said gene is a 7-keto-8-aminopelargonate synthetase gene, a 7,8-diaminopelargonate aminotransferase gene, a desthiobiotin synthetase gene, a biotin synthase gene, or a gene coding for an enzyme having activity to catalyze a reaction in a biosynthesis stage upstream to pimelyl Co-A in the biotin biosynthetic pathway.
30) A DNA fragment having the base sequence shown as SEQ ID NO: 36 or 37.
31) A DNA fragment according to any one of the above items 1, 2, 3, 7, 11, 15, 20, 24, 25, 26, 27, 28 and 29, wherein the microorganism belonging to the genus Sphingomonas is Sphingomonas paucimobilis JCM7511 or Sphingomonas sp. SC42405.
32) A vector containing a DNA fragment according to any one of the above items 1 to 31.
33) A method for preparing a vector which comprises inserting a DNA fragment according to any one of the above items 1 to 31 to a vector replicable in host cells.
34) A vector according to the above item 32, wherein a gene expression regulatory region is linked upstream to a region coding for a protein.
35) A transformant having at least one DNA fragment according to any one of the above items 1 to 31 or at least one vector according to the above item 32 or 34 introduced into a host cell.
36) A transformant according to the above item 35, wherein the host cell is a microorganism.
37) A method for preparing transformants which comprises introducing a vector according to the above item 32 or 34 into a host cell.